EP4134378A1 - Method for generating high affinity antibodies - Google Patents

Method for generating high affinity antibodies Download PDF

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EP4134378A1
EP4134378A1 EP22199396.7A EP22199396A EP4134378A1 EP 4134378 A1 EP4134378 A1 EP 4134378A1 EP 22199396 A EP22199396 A EP 22199396A EP 4134378 A1 EP4134378 A1 EP 4134378A1
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Prior art keywords
cells
antigen
minutes
antibodies
antibody
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German (de)
English (en)
French (fr)
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Gang Chen
Robert Babb
James P. Fandl
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Regeneron Pharmaceuticals Inc
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Regeneron Pharmaceuticals Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention generally relates to high-affinity antibodies. More particularly, the present invention relates to a method for selecting high affinity antibodies.
  • High affinity is a desirable attribute for many therapeutic antibodies.
  • the availability of high affinity monoclonal antibodies is crucial to the development of targeted immunotherapies.
  • very high affinity antibodies are rare and standard methods for generating antibodies are inefficient at isolating high affinity antibodies.
  • monoclonal antibodies are obtained from mouse hybridomas, which most often result from the fusion of B lymphocytes from immunized mice with murine myeloma cells.
  • the isolation of rare high affinity antibodies by hybridoma technology is not efficient because of throughput limitations on hybridoma culture.
  • Another approach to producing high affinity antibodies involves the use of display technology to produce a lead antibody candidate from a phage, yeast or mammalian library. Though direct DNA isolation from B cells expressing antibodies may be utilized, DNA libraries are expressed in cell expression systems, such as phage, yeast, or bacterial systems, then "panned" or titrated to select for the antibodies having high affinities. Display technologies can provide high-quality protein libraries, although they provide limited diversity. Consequently, in vitro mutagenesis-based affinity maturation is frequently a next step in generating high affinity antibodies derived from such libraries.
  • the present invention is based on the observation that cells expressing antibody with high affinity for an antigen of interest can be selected and enriched directly from a population of antibody-producing cells of varying affinities.
  • the antibodies are thus selected directly from primary antibody-producing cells, prior to a single-cell isolation and collection, such as using fluorescence activated cell sorting (FACS).
  • FACS fluorescence activated cell sorting
  • the invention relates to a method in which primary antibody-producing cells expressing an antigen-specific antibody are efficiently selected based on their binding properties in situ, then isolated using techniques for single-cell isolation, such as using fluorescence activated cell sorting (FACS), a high throughput screening method that can sample hundreds of millions of cells in a cell population.
  • FACS fluorescence activated cell sorting
  • the cells expressing desirable high affinity antibodies can be identified and isolated directly from all of the cells producing antibodies (rather than from screening antibody libraries following cloning steps).
  • the antibodies produced by the selected cells can then be cloned and reproduced recombinantly in host cells for direct use, thereby diminishing the number of steps taken while ensuring a higher probability of desirable antibodies.
  • the present invention relates to a method for the selection of antibodies that specifically bind an antigen of interest with high affinity.
  • the method may be used for efficient isolation of antibodies to a monomeric antigen with affinities ranging from about 0.1 pM to about 10 nM.
  • the invention relates to a method for selecting cells that express antibody exhibiting high binding affinity for an antigen of interest from a population of cells that express antibodies of different binding affinity for the antigen, the method comprising: contacting a population of primary antibody-producing cells with specificity to an antigen of interest with a low concentration of labeled antigen for a time sufficient for the labeled antigen to bind to antibody on the surface of the cells; washing the labeled antigen-bound cells with an appropriate buffer for a period of time from about 15 minutes to about 60 minutes; then isolating the antigen-bound cells.
  • the isolation step may further comprise identifying the antigen-bound cells with an antigen-binding protein comprising a label for identification.
  • the method may even further comprise contacting the cells with an antigen-binding protein comprising a fluorescent label; and using fluorescence-activated cell sorting, thus identifying single cells that have bound antigen and express an antibody that specifically binds the antigen of interest with high affinity.
  • the cell population is contacted with a low concentration of labeled antigen between about 1 nM and about 15 nM, in some embodiments between about 2 nM and about 10 nM; and in some embodiments, the concentration of biotinylated antigen is about 5 nM.
  • the cells to which labeled antigen has bound will exhibit high fluorescence intensity (FI).
  • the method further relates to a method for separating high affinity antibodies from low affinity antibodies, such as isolating the antibodies having a K D less than about 25 nM, or less than about 20 nM, or less than about 15 nM, or less than about 10 nM.
  • the invention relates to a cell selection method wherein antigen-specific cells are contacted with biotinylated antigen.
  • the method further comprises fluorescently labeled streptavidin.
  • the concentration of biotinylated antigen is between about 1 nM and about 15 nM, in some embodiments between about 2 nM and about 10 nM; in some embodiments, the concentration of biotinylated antigen is about 5 nM.
  • the method further comprises collecting the cells that exhibit high fluorescent intensity to obtain cells that express antibodies of high affinity for the antigen of interest.
  • Host cells comprising a nucleic acid molecule encoding the antibody isolated using the methods of the invention are also contemplated.
  • the method relates to a method for selecting high affinity antigen-specific antibody-producing cells from a population of cells selected from the group consisting of plasma cells, B cells, hybridomas and other recombinant cells, plasma cell myelomas, and tissue-derived cells such as spleen, lymph node, bone marrow or peripheral blood.
  • the present invention may be used for efficient production of antibodies with affinities ranging from about 0.1 pM to about 25 nM, particularly having an K D less than 10 nM.
  • Antibody and antibodies as those terms are known in the art refer to antigen binding proteins of the immune system.
  • the term antibody as referred to herein includes whole, full length antibodies having an antigen-binding region, and any fragment thereof in which the "antigen-binding portion” or “antigen-binding region” is retained, or single chains, for example, single chain variable fragment (scFv), thereof.
  • a naturally occurring "antibody” is a glycoprotein comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant (CH) region.
  • the heavy chain constant region is comprised of three domains, CH1, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant CL region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with antigen.
  • antibody-producing cells and “antibody-expressing cells” refer to cells that express antibody either naturally, i.e., as the result of B-cell activation or as the result of recombinant technology and genetic engineering.
  • the term therefore, encompasses lymphocytes of antigen-dependent B-cell lineage, including memory B-cells, plasmablasts and terminally differentiated plasma cells as well as recombinant cells such as hybridomas and non-lymphoid cells engineered to express antibodies.
  • Primary cells refers to cells grown outside of their natural environment, such as tissue cells isolated from a mammal. In certain embodiments, primary antibody-producing cells are tissue-derived, such as from spleen, lymph node, bone marrow or peripheral blood.
  • antibody-producing cells and “cells expressing antibodies” encompass cells in which the antibodies expressed are bound to or anchored in the cell membrane, i.e. cell surface antibodies, as well as cells that secrete antibody.
  • further antibody-producing cells may be derived from the starting primary antibody-producing cells or the primary antibody-producing cells selected by the methods of the invention.
  • cell lines, plasma cells, memory B-cells, hybridomas, plasma cell myelomas and recombinant antibody-expressing cells may be derived or isolated from primary antibody-producing cells prior to or following collection of antibody-producing cells expressing high-affinity antibodies.
  • primary antibody-producing cells may be fused to myeloma cells to make hybridomas, or otherwise immortalized, such as infected with a virus (e.g. EBV), or may be differentiated by cell sorting techniques based on protein markers expressed by particular B cell types. For example, selected antibody-producing cells expressing high-affinity antibodies may be sorted by FACS based on cell surface B cell markers.
  • a virus e.g. EBV
  • Binding affinity generally refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, "binding affinity” refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule for its binding partner can generally be represented by the dissociation equilibrium constant (KD or K D ).
  • K D dissociation equilibrium constant
  • M the K D value
  • higher affinity refers to antibodies that generally bind antigen stronger and/or faster and/or remain bound longer. Generally, a lower concentration (M) of antigen is needed to achieve the desired effect due to its stronger binding interaction.
  • low affinity and lower affinity is a term used to reflect a weaker binding, such as a lower ability to form an interaction and therefore a larger K D value.
  • Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily.
  • kd (sec -1 or 1/s) refers to the dissociation rate constant of a particular antibody-antigen interaction, or the dissociation rate constant of an antibody, Ig, antibody-binding fragment, or molecular interaction. This value is also referred to as the koff value.
  • ka (M-1 ⁇ sec-1 or 1/M) refers to the association rate constant of a particular antibody-antigen interaction, or the association rate constant of an antibody, Ig, antibody-binding fragment, or molecular interaction.
  • KD or "K D " (M) refers to the equilibrium dissociation constant of a particular antibody-antigen interaction, or the equilibrium dissociation constant of an antibody, Ig, antibody-binding fragment, or molecular interaction.
  • the equilibrium dissociation constant is obtained by dividing the ka with the kd.
  • Binding affinities obtained using the method are in the range of about 0.1 pM to about 10 nM as determined by surface plasmon resonance.
  • high affinity antibody refers to those antibodies having a binding affinity, expressed as K D , having a numerical value of about 0.1 pM to about 25 nM, or about 0.1 nM to about 10 nM.
  • high affinity antibodies have a measured K D about 2.5 ⁇ 10 -9 M or less, about 1 ⁇ 10 -10 M or less, or about 0.5 ⁇ 10 -10 M or less, as measured by surface plasmon resonance, e.g., BIACORE TM or solution-affinity ELISA.
  • K D of antibodies may be represented numerically either as nE-z, or as n ⁇ 10 -z , for example, 3.2E-12 is equivalent to 3.2 ⁇ 10 -12 and indicates a K D of 3.2 picomolar (pM).
  • the present invention provides a method to identify and isolate antigen-specific antibody-producing cells that express antibodies exhibiting a high binding affinity for an antigen of interest; the nucleic acids encoding these antibodies can then be cloned into host cells for mass production of the high affinity antibodies.
  • the present method avoids the use of mutation to enhance binding affinity.
  • the method also obviates the need for panning of antibody libraries following expression of such antibodies in a host cell system.
  • Immunization of mammals can be done by any methods known in the art (see, for example, E. Harlow and D. Lane - Antibodies A Laboratory Manual, Cold Spring Harbor (1988 ); Malik and Lillehoj, Antibody techniques: Academic Press, 1994, CA ).
  • Antigen may be administered directly to a mammal, without adjuvant, or with adjuvant to aid in stimulation of the immune response.
  • Adjuvants known in the art include, but are not limited to complete and incomplete Freund's adjuvant, MPL+TDM adjuvant system (Sigma), or RIBI (muramyl dipeptides) (see O'Hagan, Vaccine Adjuvant, by Human Press, 2000, NJ ). Without relying on a particular theory, adjuvant can prevent rapid dispersal of polypeptide by sequestering the antigen in a local depot, and may contain factors that can stimulate host immune response.
  • the antigen of interest is administered as a protein, protein fragment, protein-fusion or DNA plasmid that contains the antigen gene of interest and expressing the antigen of interest using the host cellular expression machinery to express the antigen polypeptide in vivo.
  • Various immunization techniques are known in the art. It is understood that the immunized mammal may be a human having been exposed to antigen and expressing humoral immunity for the antigen of interest.
  • a non-human mammal is immunized with an antigen of interest and the animal's immune response to the antigen is monitored using an antigen-specific immunoassay. Once an appropriate immune response has been achieved, antibody-producing cells are collected from the immunized animal. Antibody-producing cells are collected from a number of sources, including but not limited to spleen, lymph node, bone marrow and peripheral blood.
  • splenocytes are harvested from an immunized animal.
  • IgG + antigen-positive B cells from the immunized animals are isolated from the cell population using the methods described herein.
  • peripheral blood mononuclear cells are harvested from a human or non-human mammal known to have humoral immunity to an antigen of interest.
  • IgG + , antigen-positive B cells having the highest affinities in the antibody-producing cell population can then be isolated for further processing in accordance with the methods of the invention.
  • the harvested cells are contacted with a low concentration, for example, from about 0.1 nM to about 25 nM, or from about 1 nM to about 20 nM or from about 2 nM to about 10 nM, of monomeric antigen that is labeled, for a time sufficient for the labeled antigen to bind to antibody on the surface of the immune cells; in some embodiments, exposure of the immune cells to labeled antigen for from about 5 to about 60 minutes is suitable. In some embodiments, the low concentration is less than about 10nM.
  • the low concentration of antigen is about 9nM, about 8nM, about 7nM, about 6nM, about 5nM, about 4nM, about 3nM, about 2nM, about 1nM. In still other embodiments, the antigen concentration is 5nM. In some embodiments, the antigen concentration is less than about 1nM. In another embodiment, the antigen concentration is 1 nM. In another embodiment, the antigen concentration is less than 1nM. In other embodiments, the antigen is soluble.
  • the label is biotin, e.g., the antigen is biotinylated.
  • Antigen labels otherwise called detection molecules, enable further detection of the antigen of interest bound to the antibody-producing cells. Detection may be made by immuno-staining with an antibody specific for the label or direct staining with a reagent that binds to the label. Numerous detection kits and techniques are well-known in the art.
  • washing the cells for a period of time comprising one (1) wash about 10 minutes, or about 15 minutes, or about 20 minutes, or about 25 minutes, or about 30 minutes, or about 35 minutes, or about 40 minutes, or about 45 minutes, or about 50 minutes, or about 55 minutes, or about 60 minutes total, may be used.
  • washing the cells for a period of time comprising two (2) washes each about 5 minutes, or about 10 minutes, or about 15 minutes or about 20 minutes, or about 25 minutes, or about 30 minutes per wash, may be used. Additional washing intervals are contemplated, essentially equivalent to those described herein.
  • the antigen of interest may be found in nature as a polymer, such as a dimer
  • the antigen may be engineered to a monomer for the purposes of the invention.
  • partial denaturation of polymers, protein engineering or protein fusion techniques may be employed by the skilled artisan.
  • the dimerization domain may be mutated or deleted to disallow dimerization.
  • the antigen of interest may be utilized as a dimer or other multimeric form (i.e. tetramer, heptamer, hexamer, etc.) for immunization purposes, however the same target epitopes of the antigen of interest are employed in a monomer for the purposes of enrichment of high-affinity antibodies.
  • the cells may be detected as B cells, in particular IgG+IgM- cells (incubated with anti-B cell marker, anti-IgG or anti-Fc reagents, or the like) in the anticipation of next step single-cell isolation techniques.
  • IgG or B cell detection reagents may be incubated with the cells prior to, during, or following the incubation with antigen of interest.
  • B cell detection reagents are commercially available. (See also Huang, J. et al., 2013 Nature Protocols, 8(10):1907-1915 .)
  • the selected cells are enriched for high-affinity antibodies. It has been unexpectedly found that by way of this invention a selected pool of cells expressing antibodies was enriched by achieving a high percentage of high-affinity antibodies isolated from the selected cells, such as greater than 50% 55%, 60%, 65%, 70%, 75%, 80%, or 85% isolated antibodies, or greater than 90%, 95%, 96%, 97% or 98% isolated antibodies are high-affinity antibodies.
  • the cells are contacted with an antigen-binding protein comprising a detectable label, for example, a fluorescent label for the purposes of identifying the antigen-specific cells.
  • a detectable label for example, a fluorescent label for the purposes of identifying the antigen-specific cells.
  • a detectable label for example, a fluorescent label for the purposes of identifying the antigen-specific cells.
  • FACS fluorescence-activated cell sorting
  • Cells may be sorted and collected by alternative methods known in the art, including but not limited to manual single cell picking, limited dilution and B cell panning of adsorbed antigen, which are all well-known in the art ( Rolink, et al., 1996 J Exp Med 183:187-194 ; Lightwood, D. et al, 2006 J. Immunol. Methods 316(1-2):133-43. Epub 2006 Sep 18 ).
  • Isolated B cells may be fused with an immortal cell, such as a myeloma cell line, in order to create a hybridoma. Hybridoma techniques are well within the skill of the artisan (Harlow and Lane, 1988, supra ). Isolated B cells may be further differentiated or sorted to identify specific B cell types, such as determination by cell surface or gene expression markers.
  • an immortal cell such as a myeloma cell line
  • B cells may be cultured, fused to myeloma cells or otherwise immortalized, such as infected with a virus (e.g. EBV), in order to make the DNA more abundant, as necessary, prior extracting DNA and cloning antibody genes directly from each sorted B cell.
  • genes encoding immunoglobulin variable heavy and light chains i.e. VH, Ig V ⁇ and V ⁇
  • VH, Ig V ⁇ and V ⁇ genes encoding immunoglobulin variable heavy and light chains (i.e. VH, Ig V ⁇ and V ⁇ ) are recovered using RT-PCR of mRNA isolated from the selected antibody-producing cells, as performed using conventional techniques, for example, as described by Wang et al. (J. Immunol. Methods 244:217-225 ) and described herein.
  • Antibody genes are cloned into IgG heavy- and light-chain expression vectors and expressed via transfection of host cells.
  • the nucleic acid encoding the antibody genes are inserted into a replicable vector for further cloning (amplification of the DNA) or for expression (stably or transiently).
  • a replicable vector for further cloning (amplification of the DNA) or for expression (stably or transiently).
  • Many vectors, particularly expression vectors, are available or may be engineered to comprise appropriate regulatory elements.
  • An expression vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, and synthetic nucleic acid vectors (a nucleic acid sequence comprising a suitable set of expression control elements).
  • vectors examples include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors.
  • an antibody-encoding nucleic acid molecule is comprised in a naked DNA or RNA vector, including, for example, a linear expression element (as described in, for instance, Sykes and Johnston, Nat Biotech 12:355-59 (1997 )), a compacted nucleic acid vector (as described in for instance US6,077,835 and/or WO00/70087 ), or a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119.
  • a linear expression element as described in, for instance, Sykes and Johnston, Nat Biotech 12:355-59 (1997 )
  • a compacted nucleic acid vector as described in for instance US6,077,835 and/or WO00/70087
  • a plasmid vector such as pBR322, pUC 19/18, or pUC 118/119.
  • Such nucleic acid vectors and the usage thereof are well known in the art (see, for instance, US5,589
  • An expression vector may alternatively be a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be employed. Suitable vectors include, for example, vectors comprising constitutive or inducible promoters such as yeast alpha factor, alcohol oxidase and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987 ), and Grant et al., Methods in Enzymol 153, 516-544 (1987 )).
  • constitutive or inducible promoters such as yeast alpha factor, alcohol oxidase and PGH
  • the vector comprises a nucleic acid molecule (or gene) encoding a heavy chain of the antibody and a nucleic acid molecule encoding a light chain of the antibody, wherein the antibody is produced by the B cell selected by a method of the invention.
  • the vector utilized includes an expression vector comprising the nucleic acid molecules (genes) described wherein the nucleic acid molecule (gene) is operably linked to an expression control sequence suitable for expression in the host cell.
  • Host cells include, but are not limited to, cells of either prokaryotic or eukaryotic (generally mammalian) origin.
  • the host cell is a bacterial or yeast cell. In some embodiments, the host cell is a mammalian cell. In other embodiments, the host cell is selected from the group consisting of CHO (e.g. CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g. COS-7), stem cell, retinal cells, Vero, CV1, kidney (e.g.
  • the full-length antibody may be subsequently cloned into an appropriate vector or vectors.
  • the Fab region of an isolated antibody may be cloned into a vector or vectors in line with constant regions of any isotype for the intended purpose. Therefore, any constant region may be utilized in the construction of isolated antibodies, including IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgD, and IgE heavy chain constant regions, or chimeric heavy chain constant regions.
  • Such constant regions can be obtained from any human or animal species depending on the intended use of the antibodies.
  • antibody variable regions or Fab region may be cloned in an appropriate vector(s) for the expression of the protein in other formats, such as ScFv, diabody, etc.
  • the invention provides a mammalian host cell encoding a nucleic acid molecule comprising a high affinity antibody specific for an antigen of interest, wherein a heavy chain variable region and a light chain variable region of the antibody were isolated from a B cell expressing the antibody, and wherein the B cell was selected from a population of cells from an immunized mammal with a low concentration of the antigen in monomeric form.
  • Binding affinities and kinetic constants of the antibodies derived from cells isolated using the method of the invention are determined in accordance with methods known in the art, for example, by surface plasmon resonance. In one embodiment, measurements are conducted at 25°C on, for example, a Biacore 2000 or similar instrument. Antibodies are captured on an anti-human Fc sensor surface, and soluble monomeric protein is injected over the surface. Kinetic association (k a ) and dissociation (k d ) rate constants are determined by processing and fitting the data to a 1:1 binding model using curve fitting software.
  • Example 1 Separation of micro beads coated with a fast dissociation antibody from micro beads coated with a slow dissociation antibody by flow cytometry.
  • B cells from immunized mice express BCRs of varying affinity against an immunogen.
  • B cells from immunized mice express BCRs of varying affinity against an immunogen.
  • labeled antigen human ExTek.6His; see Lin, P. et al. PNAS USA. 1998 Jul 21; 95(15): 8829-8834
  • polystyrene micro beads catalog. number MPFc-60-5, Spherotech, Lake Forest, IL
  • mAb 12P3 or 39P2 two mouse monoclonal antibodies against human Tie2 protein.
  • Polystyrene beads conjugated with polyclonal anti-mFc capture antibodies were incubated with either 12P3 or 39P2 overnight. The next day, the beads were washed with PBS to remove unbound 12P3 and 39P2 antibodies and then incubated with 5 nM biotin-hExTek for one hour. Each sample was then divided into two aliquots. The beads in all samples were pelleted by centrifugation at 1,000 g for 4 minutes.
  • Fig. 1 shows an overlay of flow cytometry data for the two Tie2 mAb- coated beads, either without a wash step or washed twice, before incubation with PE-streptavidin. There was a clear separation in their profiles, when washed twice for 10 min each. The clear separation of the FACS profiles indicates that the 39P2-coated beads could be isolated from a mixed beads population comprised of the 39P2 beads and 12P3 beads.
  • B cells expressing surface-anchored antibodies with fast or slow dissociation constants can be separated by using 5 nM of labeled antigen to label the B cells and incorporating a subsequent wash step before the addition of a secondary detection reagent such as phycoerythrin (PE)-streptavidin.
  • a secondary detection reagent such as phycoerythrin (PE)-streptavidin.
  • Example 2 Separation of hybridoma cells expressing a low affinity antibody from hybridoma cells expressing a high affinity antibody by flow cytometry.
  • the 39P2 cell line contained two populations of cells with different levels of surface IgG expression. These two hybridoma cell lines were stained with 5 nM biotin-hExTek for one hour. The two stained cell populations were each divided into three aliquots. One aliquot for each cell line sample was given excess PE-streptavidin without the removal of biotin-hExTek and incubated for 30 minutes. In parallel, the other aliquots for each cell line sample were washed with excess PBS for either 20 minutes or 40 minutes, then stained with PE-streptavidin for 30 minutes.
  • PE fluorescence of all cell samples was analyzed by FACS ( Fig. 2 ).
  • the 39P2 cell line was comprised of two populations of cells with different levels of PE fluorescence on FACS: PE high and PE low , due to differing levels of surface IgG expression.
  • the mean fluorescence intensity of the PE high 39P2 cells did not change significantly when the cells were washed for either 20 min or 40 min prior to staining with PE-streptavidin.
  • the PE fluorescence of the stained 12P3 cells mostly overlaps with the PE fluorescence of the PE high 39P2 cells.
  • PE fluorescence of the 12P3 cells that were washed for either 20 min or 40 min following incubation with biotin-hExTek but prior to staining with PE-streptavidin was significantly less than the PE fluorescence of the PE high 39P2 cells, such that these two cell populations were well separated on FACS.
  • the separation of the FACS histograms of the cell populations enables isolation of the PE high 39P2 cells from a mixture of PE high 39P2 and 12P3 cells by sorting for high PE cells on a flow cytometer.
  • VELOCIMMUNE ® mice were immunized with a purified protein comprised of Tie2 extracellular domain and human Fc domain (Tie2-hFc). This protein is a dimer due to Fc:Fc association. After three injections of Tie2-hFc to boost immune response, mice were sacrificed and splenocytes were harvested. The splenocytes were pooled and divided into two aliquots. Cells in one aliquot were stained with 5 nM biotinylated Tie2-hFc protein and FITC-anti-mFc for 1 hour.
  • the other half of the splenocyte pool was stained with 5 nM biotinylated ExTek and FITC-anti-mFc for 1 hour.
  • the stained cells were washed for 10 minutes with PBS, then stained with SA-PE for one hour.
  • the stained cells were washed once with PBS and were analyzed by flow cytometry on a MoFlo (Cytomation).
  • Single IgG positive and antigen positive B cells were sorted into separate wells on a 96-well plate.
  • RT-PCR of antibody genes from these B cells was performed according to a method described by Wang and Stollar (Journal of Immunological Methods 2000; 244 : 217-225 ). Briefly, cDNAs for each single B cell were synthesized via reverse transcriptase (RT) reaction (Superscript TM III, Invitrogen). Each resulting RT product was then split and transferred into two corresponding wells on two 96-well plates.
  • RT reverse transcriptase
  • One set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific for human IgG heavy chain variable region leader sequence and a 3' primer specific for mouse heavy chain constant region, to form an amplicon.
  • the amplicon was then amplified again by PCR using a 5' degenerate primer set specific for framework 1 of human IgG heavy chain variable region sequence and a 3' degenerate primer set specific for framework 4 of human IgG heavy chain variable region sequence.
  • the other set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific for human kappa light chain variable region leader sequence and a 3' primer specific for mouse kappa light chain constant region to form an amplicon.
  • the amplicon was then amplified again by PCR using a 5' degenerate primer set specific for framework 1 of human kappa light chain variable region sequence and a 3' degenerate primer set specific for framework 4 of human kappa light chain variable region sequence.
  • the heavy chain and light chain PCR products were cloned into antibody vectors containing human IgG1 heavy chain constant region and kappa light chain constant region, respectively.
  • Recombinant hIgG1 antibodies were produced by transient transfection of CHO K1 cells. Dissociation constants of the binding between ExTek protein and 61 Tie2-specific antibodies present in these culture supernatants were determined on Biacore.
  • a protein immunogen comprising the human Erb3 extracellular domain fused to mouse Fc (Erb3.mFc) was produced in CHO cells and purified.
  • the Erb3.mFc protein was administered directly to VELOCIMMUNE ® mice comprising DNA encoding human Immunoglobulin heavy and kappa light chain variable regions (Regeneron Pharmaceuticals, Inc., US patent 6,596,541 ).
  • the antibody immune response was monitored by an Erb3-specific immunoassay. When a desired immune response was achieved splenocytes were harvested. Red blood cells were removed by lysis followed by pelleting the harvested splenocytes.
  • Resuspended splenocytes were first incubated with FITC-anti-mFc and 5 nM biotinylated monomeric Erb3 protein (Erb3.mmh) for 1 hour. The stained cells were washed for 10 minutes with PBS, then stained with SA-PE for one hour. The stained cells were washed once with PBS and were analyzed by flow cytometry on a MoFlo (Cytomation).
  • Each IgG positive, antigen-positive B cell was sorted and plated into a separate well on a 384-well plate.
  • RT-PCR of antibody genes from these B cells was performed according to a method described by Wang and Stollar (Journal of Immunological Methods 2000; 244 : 217-225 ). Briefly, cDNAs for each single B cell were synthesized via reverse transcriptase (RT) reaction (Superscript TM III, Invitrogen). Each resulting RT product was then split and transferred into two corresponding wells on two 96-well plates.
  • RT reverse transcriptase
  • One set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific for human IgG heavy chain variable region leader sequence and a 3' primer specific for mouse heavy chain constant region, to form an amplicon.
  • the amplicon was then amplified again by PCR using a 5' degenerate primer set specific for framework 1 of human IgG heavy chain variable region sequence and a 3' degenerate primer set specific for framework 4 of human IgG heavy chain variable region sequence.
  • the other set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific for human kappa light chain variable region leader sequence and a 3' primer specific for mouse kappa light chain constant region to form an amplicon.
  • the amplicon was then amplified again by PCR using a 5' degenerate primer set specific for framework 1 of human kappa light chain variable region sequence and a 3' degenerate primer set specific for framework 4 of human kappa light chain variable region sequence.
  • the heavy chain and light chain PCR products were cloned into antibody vectors containing human IgG1 heavy chain constant region and kappa light chain constant region, respectively.
  • the light chain expression plasmid has a loxP site and lox2272 site flanking the light chain expression cassette, and the heavy chain expression plasmid has a lox2272 site and a lox511 site flanking the heavy chain expression cassettes.
  • Plasmids having either human heavy chain variable region sequence or human light chain variable region sequence were isolated from transformed E . coli cultures. Purified plasmids having a human heavy chain variable region sequence and plasmids having a human light chain variable region sequence from the same B cell were then combined and transfected into a modified EESYR ® CHO host cell. Since the host CHO cell has, from 5' to 3', a loxP site, an eCFP, a lox2272 site, DsRed, and a lox511 site at the EESYR ® locus, the host CHO cell can be isolated by flow cytometry as a blue-positive, red-positive, and green-negative cell.
  • plasmids transiently expressing Cre were also transfected into the modified EESYR ® CHO host cell.
  • the transfected EESYR ® CHO cells were sorted by flow cytometry. Proper recombinants that show blue-negative, red-negative, and green-positive cells were isolated and stable recombinant antibody expressing CHO cell lines were established from the isolated proper recombinants.
  • Figure 4 shows that among the 148 anti-Erb3 antibodies isolated by selecting B cells with 5 nM biotin-Erb3.mmh protein, 78 Abs bound Erb3.mmh with K D s of less than 1 nM, 57 Abs bound Erb3.mmh with K D s between 1 nM to 10 nM, 12 Abs bound Erb3.mmh with K D s between 10 nM to 100 nM, and 1 Ab bound Erb3.mmh with K D between 100 nM to 1 ⁇ M.
  • Anti-Erb3 antibodies with less than 10 nM K D significantly out-numbered antibodies with greater than 10 nM K D , demonstrating that selecting and isolating B cells that bound 5 nM biotin-Erb3.mmh enriched for antibodies with high affinity (less than 10 nM K D ).
  • anti-Erb3 antibodies with the best affinity against Erb3.mmh were produced from stable EESYR ® CHO cells and purified by protein A affinity chromatography.
  • Figure 5 shows that among the 63 anti-NGF antibodies isolated by selecting B cells with 5 nM biotin-NGF, 55 Abs bound NGF with KDs of less than 1 nM, 7 Abs bound NGF with KDs between 1 nM to 10 nM, and 1 Ab bound NGF with KDs between 10 nM to 100 nM.
  • Anti-NGF antibodies with less than 10 nM KD significantly out-numbered antibodies with greater than 10 nM KD, demonstrating that selecting and isolating B cells that bound 5 nM biotin-NGF enriched for antibodies with high affinity (less than 10 nM KD).
  • Figure 6 shows that among the 648 anti-Activin A antibodies isolated by selecting B cells with 1 nM biotin-Activin A, 212 Abs bound Activin A with KDs of less than 1 nM, 416 Abs bound Activin A with KDs between 1 nM to 10 nM, and 20 Abs bound Activin A with KDs between 10 nM to 100 nM.
  • Anti- Activin A antibodies with less than 10 nM KD significantly out-numbered antibodies with greater than 10 nM KD, demonstrating that selecting and isolating B cells that bound 1 nM biotin-Activin A enriched for antibodies with high affinity (less than 10 nM KD).

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